Proper function of cytokine signaling pathways is critical for immunoregulation, hematopoiesis, and cytokine- directed inflammation and growth. Most signals from type I and type II cytokines are mediated by the Janus kinase (Jak) family of non-receptor tyrosine kinases. Our plan is to discover the molecular basis for cytoplasmic signal transduction from the interleukin-2 family cytokines through Jak3. We will use structural biology to provide the first description of Jak interactions with cytokine receptor cytoplasmic tails, to investigate Jak kinase domain specificity, regulation and inhibition, and to describe the role of the PERM domain in kinase activation. Specifically, we will focus our work in three distinct areas. In Aim 1 we will determine crystal structures of the kinase domain of Jak3. Modulation of the immune response by suppression of Jak3 kinase activity has clinical precedence. We will determine the mode of binding for Jak3 tyrosine kinase inhibitors and aid the discovery of further inhibitors that may be useful as immunomodulatory and anti-proliferative therapies. We will also discover the mode of Jak3 binding to a Jak3-preferred substrate and discover the inactive conformation of the kinase. In Aim 2, we will determine the structural basis for the almost exclusive Jak3 association with the interleukin-2 receptor gamma subunit. For this we will solve crystal structures that describe the interaction of the Jak3 PERM domain with peptides from the interleukin-2 receptor gamma subunit and investigate their binding affinities. Finally, following our structural studies on the kinase and PERM domains of Jak3, in Aim 3 we will determine the structural basis for Jak3 kinase domain association with the Jak3 FERM domain. We will also test the role of this interface as a suggested Jak3 active state stabilizing mechanism. These studies will directly impact healthcare stimulating the development of improved immunomodulatory and anti-proliferative therapies and by enhancing molecular-level understanding of cytokine signaling pathways.